Pediat. Res. 14: 254-258 (1980) liposome phospholipase A lung respiratory dis- phosphatidylcholine tress syndrome surfactant The Action of Lung Lysosomal Phospholipases on Dipalmitoyl Phosphatidylcholine and its Significance for the Synthesis of Pulmonary Surfactant M. F. Heath"" and W. Jacobson S~rangewaysResearch Laboratory, Cambridge, United Kingdom Summary numerous lysosomal hydrolases (16). The origin and purpose of this lysosomal involvement is unsure, but it seems likely that Subcellular fractions were prepared from rabbit lung and char- lysos6mes take up newly synthesized surfactant by an autophagic acterized by marker enzyme assays. Phospholi~asesA of lysoso- mechanism and process it to the characteristic lamellar bodies ma1 type (active at pH 4.0 in the absence of Ca +) show a peak of before it is released from the cell by exocytosis (16, 34, 36). specific activity in fraction P3 (13000 x g; 20 min; pellet). About The lysosomal hydrolases present in lamellar bodies include 70% of the phospholipase A activity of P3 can be obtained in a two enzymes capable of catabolizing the phosphatidylcholine of soluble extract. The rates of hydrolysis of dipalmitoyl (saturated) surfactant, phospholipases A, (EC 3.1.1.32) and A2 (EC 3.1.1.4) and unsaturated -phosphatidylcholine - molecules, presented to en- (1 7). Phospholipase A, hydrolyzes the fatty acid residue at position zyme preparations in liposomes of varying composition, are shown I of phosphatidylcholine and other phospholipids; phospholipase in Tables 3 to 5. Although- the rate for unsaturated molecules is AP removes the acid at the 2-position. The lysosomal forms of unaffected by liposome composition, saturated molecules are hy- these enzymes have maximum activity near pH 4, are inhibited by drolyzed significantly faster in 5 mol % dipalmitoyl phosphatidyl- Ca2', and require the phospholipid for hydrolysis to be in the choline liposomes than in 95 mol %. With enzyme preparation I form of a bilayer, such as the lamellae of the lung inclusion bodies (60% A,; 40% Az) the increase was dfold (P< 0.01). Preparations or the liposomes (lipid vesicles) used as the assay substrate. I and I1 hydrolyzed both kinds of molecules at similar rates in 50 Aside from their catabolic role, these enzymes could function in mol % dipalmitoyl phosphatidylcholine liposomes, but preparation the normal process of surfactant biosynthesis. The lysosomal 111, with a higher proportion of A2 (m),gave a notably lower phospholipase A2 of the lamellar bodies in particular may be rate of hydrolysis of saturated molecules. Phosphatidylglycerol(10 concerned with the remodeling of unsaturated phosphatidylcho- mol %) also decreased the rate of hydrolysis of saturated mole- line into the dipalmitoyl species by removing the unsaturated fatty cules. These results are discussed in relation to fluidity of the acid from the 2-position so that it may be replaced with a palmitoyl liposomes and to the functions of the lysosomal-type phospholi- residue (37). pases A of the lamellar inclusion bodies of the lung. Under normal conditions, the dipalmitoyl phosphatidylcholine is not degraded by these enzymes, but under abnormal conditions, Speculation the situation could arise where surfactant is synthesized at a normal rate, but is broken down intracellularly by phospholipases In lamellar bodies, phospholipase Az will preferentially hydro- before it can be released to the alveolar space. This paper describes lyze unsaturated phosphatidylcholine, yielding a lysolipid which the preparation of soluble lysosomal phospholipases from rabbit can be reacylated to produce dipalmitoyl phosphatidylcholine; lung, gives an analysis of their action on dipalmitoyl and unsatu- phospholipase Al will require further inhibition, possibly provided rated phosphatidylcholines presented to the enzymes as liposomes by the phosphatidylglycerol of surfactant. Failure of A2 action or of mixed composition, and discusses the relevance of these findings failure of the inhibition of A1 would be expected to lead to a to the roles of phospholipases A in the normal and pathologic deficiency of surfactant and thus to the respiratory distress syn- metabolism of pulmonary surfactant. drome in the newborn. MATERIALS AND METHODS Pulmonary surfactant, the agent that maintains the stability of PREPARATION OF A LYSOSOMAL FRACTION FROM RABBIT LUNG the alveoli of the lung against collapse, is a mixture of lipids with a small amount of protein. The principal lipid, comprising at least Lungs from New Zealand White rabbits were homogenized in 40% of the total, is 1,2-dipalmitoyl phosphatidylcholine (15, 22). cold 0.25 M sucrose in 50 mM Tris-HCl (pH 7) (10 ml/g) for 2 Most mammalian phosphatidylcholines have at least one unsatu- x 30 sec with a Silverson homogenizer (Silverson Machines Ltd., rated fatty acid residue (at position 2), but this major component London, United Kingdom), and the homogenate was filtered of surfactant has two saturated acids. This unusual composition is through four layers of muslin. A scheme of differential centrifu- necessary for its surfactant function (40). The respiratory distress gation was then applied as follows (see Table 1): 600 x g, 10 min syndrome of the newborn (hyaline membrane disease) results to yield pellet PI consisting of cell debris and nuclei; 5000 x g, 20 from a deficiency of pulmonary surfactant (4, 13, 20). min giving P2, mitochondria1 fraction; 13000 x g, 20 min giving The source of the extracellular surfactant is to be found in the P3, lysosomal fraction; 40,000 x g, 30 min giving P4, microsomal lamellar inclusion bodies of the cuboidal (type 11) pneumonocytes fraction, and the final supernatant, S. All pellets were resuspended of the alveolar epithelium (4, 9, 13, 20, 21)., Cytochemical< studies in 1 mM disodium EDTA in I mM NaHCOa. All fractions and a on lung sections and enzymk assays performed on isolated lamellar sample of the homogenate were subjected to five cycles of freezing body preparations have shown the presence in these inclusions of and thawing and then dialyzed overnight against 1 mM EDTA: 1 ACTION OF LUNG LYSOSOMAL PHOSPHOLIPASES 255 mM NaHC03. These fractions were analyzed for protein content West Germany), the latter diluted to 88 Ci/mole with Sigma type and characterized by assays of marker enzymes. 111-E phosphatidylcholine (from egg). These preparations were then diluted to 1.0 Ci/mole with either Sigma type 111-E phos- ANALYSIS OF LUNG FRACTIONS phatidylcholine or Sigma synthetic 1.2-dipalmitoyl phosphatidyl- choline or a mixture of the two to produce substrates with different Protein was estimated by the procedure of Lowry et al. (25) as proportions of saturated and unsaturated phospholipid, namely 5, modified by Campbell and Sargent (7) with bovine serum albumin 40, 50, 60 and 95 mol % dipalmitoyl phosphatidylcholine. The as standard (Armour Pharmaceutical Co., Chicago, IL). Lysoso- effect of phosphatidylglycerol on the hydrolysis of saturated and ma1 acid phosphatase (EC 3.1.3.2) was determined as that activity unsaturated phosphatidylcholines was investigated on mixtures of sensitive to inhibition by tartrate (see Ref. 3). with 4-nitrophenyl 9 parts of 50 mol % dipalmitoyl phosphatidylcholine with I part phosphate as substrate (Sigma Technical Bulletin 104, Sigma (mole/mole) of phosphatidylglycerol (prepared from egg phos- London Chemical Co. Ltd., Poole, Dorset, United Kin). Cathep- phatidylcholine by Lipid Products. Redhill, Surrey, United King- sin D (EC 3.4.23.5) was assayed according to Barrett (2) with dom). Liposomes of these substrates were prepared at a phospho- bovine hemoglobin (gift of Dr. A. J. Barrett) as substrate. Total lipid concentration of 0. l mM in 80 mM sodium acetate (pH 4.0) arylsulphatase (EC 3.1.6.1) was determined by the method of containing 10 mM disodium EDTA, as follows. The lipids were Worwood el al. (42) which yields a combined value for arylsul- dried from chloroform solution in a stream of N2. The buffer (N2 phatases A and B. The substrate was 2-hydroxy-5-nitrophenyl flushed) was added, and the tube was warmed at 45°C for 5 min. sulphate (Sigma London Chemical Co. Ltd.). Succinate dehydro- The contents of the tube were mixed by vortexing and then genase (EC 1.3.99.1) was assayed according to Pennington (29) as ultrasonicated for 1 min at 1.5 A in an MSE Ultrasonic Power modified by Porteous and Clark (31). NADPH cytochrome re- Unit. After rewarming to 45OC, the substrate was dispensed in 0.1- ductase (EC 1.6.2.4) was assayed with cytochrome c (Sigma Lon- ml aliquots to tubes at 37°C. The reaction was started by the don Chemical Co. Ltd.) as acceptor (30). Lactate dehydrogenase addition of 0.1 ml of enzyme preparation. (EC 1.1.1.27) was determined according to Sigma Technical Bul- Incubation proceeded for 1 hr at 37°C with continuous shaking, letin 340-UV. and then the reaction was stopped by the addition of 0.7 ml of PREPARATION OF EXTRACT CONTAINING LYSOSOMAL chloroform: methanol (l:2, v/v), and the lipids were extracted by PHOSPHOLIPASES the method of Bligh and Dyer (5). Fatty acids and phospholipids were separated by TLC in hexane:diethyl ether:acetic acid (69:29: After freezing and thawing and dialysis of fraction P3 from 2, V/V)(12) on silica gel G (Eastman Chromagram 13191 from rabbit lung, soluble phospholipases A were recovered by centrif- Kodak Ltd., Liverpool, United Kingdom). The spots were excised, ugation at 90000 x g for 2 hr. The soluble extract of P3 was and the radioactivity was measured in toluene scintillant (3.4 g/ dialyzed against 20 mM Tris-HCI (pH 7.5), applied to a column liter 2,5-diphenyloxazole and 0.05 g/liter 1,4-bis-(4-methyl-5- (1.5 x 32 cm) of Ultrogel AcA54 (LKB Instruments Ltd., Croydon. phenyloxazolyl-2)-benzene, both from Sigma London Chemical United Kingdom), and eluted with this buffer. Fractions of 1 ml Co.